Cellular Markers of Muscle Atrophy in
Chronic Obstructive Pulmonary Disease
Pamela J. Plant1, Dina Brooks2, Marie Faughnan1, Tanya Bayley1, James Bain3, Lianne Singer4, Judy Correa1,
Dawn Pearce5, Matthew Binnie1, and Jane Batt1
1
Department of Medicine and 5Department of Radiology and St. Michaels Hospital, University of Toronto, Toronto, Ontario, Canada;
2
Department of Physical Therapy, University of Toronto and Toronto Rehab Institute, Toronto, Ontario, Canada; 3Department of Surgery,
McMaster University, Hamilton, Ontario, Canada; and 4Department of Medicine, Toronto General Hospital, University Health Network,
University of Toronto, Toronto, Ontario, Canada
Skeletal muscle atrophy in individuals with advanced chronic ob-
structive pulmonary disease (COPD) is associated with diminished
quality of life, increased health resource use, and worsened survival.
Muscle wasting results from an imbalance between protein degra-
dation and synthesis, and is enhanced by decreased regenerative
repair. We investigated the activation of cellular signaling networks
known to mediate muscle atrophy and regulate muscle regenerative
capacity in rodent models, in individuals with COPD (FEV1 , 50%
predicted). Nine patients with COPD and nine control individuals
were studied. Quadriceps femoris muscle isometric contractile force
and cross-sectional area were confirmed to be significantly smaller in
the patients with COPD compared with control subjects. The vastus
lateralis muscle was biopsied and muscle transcript and/or protein
levels of key components of ubiquitin-mediated proteolytic systems
(MuRF1, atrogin-1, Nedd4), inflammatory mediators (IkBa, NF-
kBp65/p50), AKT network (AKT, GSK3b, p70S6 kinase), medi-
ators of autophagy (beclin-1, LC3), and myogenesis (myogenin,
MyoD, Myf5, myostatin) were determined. Atrogin-1 and Nedd4,
two ligases regulating ubiquitin-mediated protein degradation and
myostatin, a negative regulator of muscle growth, were significantly
increased in the muscle of patients with COPD. MuRF1, Myf5,
myogenin, and MyoD were not differentially expressed. There were
no differences in the level of phosphorylation of AKT, GSK3b,
p70S6kinase, or IkBa, activation of NF-kBp65 or NF-kBp50, or level
of expression of beclin-1 or LC3, suggesting that AKT signaling was
not down-regulated and the NF-kB inflammatory pathway and
autophagy were not activated in the COPD muscle. We conclude
that muscle atrophy associated with COPD results from the re-
cruitment of specific regulators of ubiquitin-mediated proteolytic
pathways and inhibition of muscle growth.
Keywords: vastus lateralis; ubiquitin ligase; myostatin; neural precursor
cellexpressed developmentally down-regulated 4; atrogin-1
Skeletal muscle atrophy is a critical phenomenon that occurs in
individuals with chronic obstructive pulmonary disease
(COPD). It is associated with diminished exercise capacity (1)
and quality of life (2), increased health resource use and health
care costs (3), and is a powerful negative predictor of survival
(4, 5). Loss of skeletal muscle mass results from decreased
muscle protein synthesis and increased proteolysis, and this may
(Received in original form October 8, 2008 and in final form May 8, 2009)
This work was supported by Canadian Institutes of Health Research New
Investigator Awards (D.B. and J.B.), a Keenan Foundation Fellowship, St Michaels
Hospital (P.P.), and by an Ontario Thoracic Society Grant-in-Aid Award ( J.B., D.B.,
and M.F.).
Correspondence and requests for reprints should be addressed to Jane Batt,
M.D., Ph.D., Room 7344, Medical Sciences Building, 1 Kings College Circle,
University of Toronto, Toronto, ON, M5S 1A8 Canada. E-mail: jane.batt@
utoronto.ca
Am J Respir Cell Mol Biol Vol 42. pp 461471, 2010
Originally Published in Press as DOI: 10.1165/rcmb.2008-0382OC on June 11, 2009
Internet address: www.atsjournals.org
be enhanced by decreased regenerative capacity of the muscle
(reviewed in References 68). The molecular mechanisms un-
derlying skeletal muscle atrophy have recently begun to be
elucidated through in vitro experimentation in myotube cultures
and in rodent models of disease.
Although muscle proteolysis is mediated via the coordinated
efforts of several cellular networks, including, for example,
oxidative stress responses, activation of calpains, and non-
lysosomal proteases, it is the ubiquitinproteasome pathway
that predominates (6, 812). The ubiquitin ligases, atrogin-1,
muscle-specific RING finger protein (MuRF)1, and neural
precursor cellexpressed developmentally down-regulated
(Nedd)4, key enzymes regulating ubiquitin-mediated protein
degradation, are variably up-regulated in multiple rodent
models of skeletal muscle atrophy, ranging from traumatic
denervation to chronic metabolic diseases, such as cancer and
diabetes (1317). More recently, activation of the autophagic/
lysosomal system has also been recognized to contribute to
muscle atrophy and, at least in vitro, autophagy seems to play
a substantial role in the loss of muscle protein (18, 19).
In muscle atrophy associated with generalized inflammatory
states (20, 21), the inflammatory cytokine TNF-a causes muscle
proteolysis and inhibits muscle regenerative capacity by in-
ducing oxidative stress, and destabilizing MyoD (20, 22)
through NF-kB activation. MyoD is a muscle transcription fac-
tor essential to myogenesis and muscle regeneration. NF-kB
activation also mediates expression of the ubiquitin ligase,
MuRF1, thus enhancing proteasome-mediated protein degra-
dation (23, 24).
It is also known that the engagement of muscle atrophy
signaling networks results in the concurrent down-regulation of
the signaling pathways that induce muscle hypertrophy. Acti-
vation of the AKT (protein kinase B) pathway, and signaling
through its downstream targets, such as glycogen synthase
kinase (GSK)-3b and p70S6 kinase (70-kD ribosomal protein
S6 kinase), results in muscle hypertrophy (8, 25). This pathway
is down-regulated/deactivated in models of muscle wasting,
demonstrating a key finding that there is a reciprocal link
between the muscle hypertrophic and atrophic signaling net-
works (8, 25, 26).
The muscle regulatory transcription factors, myogenin and
myogenic factor (Myf)5, which induce myoblast differentiation
and muscle regeneration and myostatin, a negative regulator of
muscle growth, can also influence muscle mass, as demonstrated
in genetic rodent models (reviewed in References 7, 27).
Few studies have assessed cellular signaling of muscle
atrophy in humans, and thus the relevance of these networks
and effectors of atrophy in human disease is not clear. In
addition, in the myriad of signaling networks activated to induce
muscle atrophy in rodent models, there is some specificity of
pathway recruitment to the atrophy model assessed. Given this
variability, we need to determine which cellular signaling
462 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
networks are active in contributing to the clinically significant
muscle atrophy of COPD. This will provide future targets for
pharmacologic manipulation, with the goal of improving quality
of life, functional status, and, potentially, survival in COPD.
Thus, in this study, we sought to identify the cellular signaling
pathways activated in atrophied skeletal muscle of patients with
severe COPD by measuring mRNA transcript and/or protein
expression levels of key signaling network components in
biopsies of the quadriceps muscles.
MATERIALS AND METHODS
Patient and Control Populations
Patients with COPD were recruited from respirology clinics at
St. Michaels Hospital (SMH) and the Toronto General Hospital,
University of Toronto. Patients were deemed eligible if they had
a physician diagnosis of COPD, as defined by the Canadian Thoracic
Society guidelines (28), and severe disease (FEV1 , 50% predicted).
Exclusion criteria included: (1) the use of systemic glucocorticoid
therapy in the preceding 90 days or the chronic use of medication that
causes muscle atrophy; (2) comorbidities associated with muscle
atrophy (infection, diabetes, end-stage renal disease on dialysis, active
cancer); (4) COPD exacerbation or hospitalization in the previous
3 months; (5) HIV, hepatitis B, or hepatitis C infection; (6) age greater
than 75 years; and (8) institutionalized care or required assistance for
activities of daily living.
A healthy age-matched control population was drawn from volun-
teer participants registered with the SMH Healthy Lung Database and
through advertisements that were placed throughout the hospitals.
These individuals had no known lung disease and did not fulfill any of
the study exclusion criteria. The study protocol was approved by the
research ethics boards at Toronto General Hospital and SMH. All
participating subjects provided informed consent.
Function and Quality of Life Measurements
Pulmonary disease severity and degree of impairment and disability
were characterized using pulmonary function testing, BODE score, and
6-minute walk distance as outlined briefly here.
Pulmonary function testing. Spirometry was performed in
accordance with recommended techniques detailed by the American
Thoracic Society guidelines (29). The primary parameters of assess-
ment were FEV1, FVC, and the FEV1:FVC ratio.
The 6-minute walk test. The 6-minute walk test was used to
assess functional exercise capacity, according to American Thoracic
Society established criteria (30, 31). We applied standardized instruc-
tion and encouragement. At baseline, the patients and control subjects
completed at least two training walks to account for learning effect.
BODE score. The BODE score incorporates body mass index
(BMI), FEV1% predicted, score on the modified Medical Research
Council dyspnea scale, and 6-minute walk distance to estimate survival
in COPD (32). The BODE score was calculated for all participants.
Assessment of Body Composition, Muscle Mass, and Strength
Muscle mass and body composition were determined using clinical
measures (BMI, sum of five skin folds, circumferential waist girth), and
the midthigh quadriceps femoris muscle cross-sectional area (CSA)
was determined as previously described (33). Briefly, computed
tomography imaging of the right thigh, halfway between the pubic
symphysis and the inferior condyle of the femur, using a Light Speed
QXi 4 slice helical scanner (General Electric, Milwaukee, WI), was
performed with the subject in the supine position. Each image was 10
to 20 mm thick, and the muscle identified as tissue with a density of 40
to 100 Hounsfield units. Images were analyzed and the CSA of muscle
determined by a single investigator, blinded as to the categorization of
each test subject.
Muscle strength was determined using isometric contractile strength
testing of the quadriceps femoris muscle. Isometric contraction of the
quadriceps femoris was measured with the use of a strain gauge, with the
subject seated on a chair extending the knee against a stationary metal
bar, as previously described (34, 35). Isometric contraction is reflective
of the amount of time a muscle group can perform and exercise.
Vastus Lateralis Percutaneous Biopsy
Biopsy of the vastus lateralis muscle was performed under local
anesthetic using a modified Bergstrom needle, as previously described
(36). Briefly, under sterile conditions, the skin and subcutaneous tissue
were anesthetized, and a small incision was made in the outer fascial
layer with a scalpel blade. The Bergstrom needle was advanced through
the incision roughly 1 cm into the muscle, suction was applied as the
trochar was advanced, and several pieces of muscle tissue were obtained.
The needle was withdrawn under counter pressure, the skin closed with
a single suture, and a pressure dressing applied. Extracted tissue was
snap frozen in liquid nitrogen for RNA and protein extraction.
RNA Extraction and Real-Time RT-PCR
Real-time RT PCR was used to assess expression levels of transcripts
for the ubiquitin ligases, atrogin-1, MuRF1, and the myogenic regula-
tory factors (MRFs), myogenin, Myf5, and myostatin (an inhibitor of
muscle growth), and mediators of autophagy, beclin-1 and autophagy-
related protein LC (LC)-3. Transcript levels were assessed as opposed
to protein levels, because good commercial antibodies were not avail-
able and/or the limited amount of protein available necessitated that
mRNA levels be determined. These genes do not produce proteins that
require phosphorylation for activation, and determination of the level
of expression of the transcripts was therefore deemed to be adequate.
Tissue snap-frozen in liquid nitrogen was homogenized in Trizol
(1 ml/mg tissue; Life Technologies, Burlington, ON, Canada) to isolate
total muscle RNA, per the manufacturers recommendations. RNA
quality was assessed with agarose gel electrophoresis and the Agilent
2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and quanti-
fied by absorption spectrophotometry at 260 and 280 nm.
To remove any contaminating genomic DNA, the muscle total
RNA samples were treated with DNA-free (Ambion, Austin, TX), per
the manufacturers instructions. cDNA was then generated from the
total RNA from each patient and control subject in a first-strand
synthesis reaction using Superscript II Reverse Transcriptase (Invitro-
gen, Burlington, ON, Canada) at 428C for 2 hours, according to the
manufacturers instructions. Relative quantitative real-time RT-PCR
was subsequently performed with an Applied Biosystems 7900 system
and SYBR Green Master Mix (Applied Biosystems, Foster City, CA)
for atrogin-1, MuRF1, myogenin, Myf5, myostatin, beclin-1, and
microtubule-associated protein 1A/1B-light chain 3 (LC3). Primers
were designed using Primer Express software (Applied Biosystems)
(Table 1).
For each gene, real-time PCR was performed in triplicate wells on
cDNA generated from the reverse transcription of 10 ng of total RNA.
Negative controls for each gene included no template (water) and no
reverse transcriptase (10 ng RNA). The PCR amplification consisted of
10-minute denaturation, followed by 40 cycles of amplification (15 s at
958C, 60 s at 608C). After amplification, amplicons were melted and the
resulting dissociation curve assessed to ensure a single product.
Aliquots (10 ml) of all products were run on DNA polyacrylamide
gels to ensure the presence of a single amplicon of the expected size.
The real-time experiments were repeated twice for all genes and all
subjects. The expression level of each transcript for each individual
(patient or control subject) was determined relative to a pooled RNA
reference generated in the laboratory, using the relative quantification
(DDCT) technique, according to Applied Biosystems instructions. Two
housekeeping genestyrosine 3 monooxygenase/tryptophan 5 mon-
oxygenase (YWHAZ), or hydroxymethylbilane synthetase (HMBS)
were used. Primer amplification efficiencies were equal for all genes
tested and both housekeeping genes (data not shown).
Protein Extraction and Western Blotting
Proteins were extracted by homogenizing the frozen muscle in lysis
buffer (5 mM Tris-HCL [pH 8.0], 1 mM EDTA, 1 mM EGTA, 1 mM
b-mercaptoethanol, 1% glycerol with 1 mM PMSF, 10 mg/ml leupeptin,
10 mg/ml aprotinin, 1 mM orthovanadate) with a Polytron PTE 1200E
homogenizer (Kinematica, Lucerne, Switzerland) in three 30-second
pulses, and homogenates were cleared by centrifuging at 1,600 3 g for
Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD 463

TABLE 1. REAL-TIME RT-PCR PRIMER SEQUENCES

Gene Forward Primer Reverse Primer

HMBS tgc aac ggc gga aga aaa agc tgg ctc ttg cgg gta c
YWHAZ gca atg atg tac tgt ctc ttt tgg aa taa cgg tag taa tct cct ttc att ttc a
Atrogin-1 gca cgt gct cag cga aga atc tgc cgc tcg gag aag t
MuRF1 tcc agc aga cac tga acc aga a tcc att ttg cac caa tgt aga aa
Myf5 agg tca acc agg ctt tcg aa gat gta gcg gat ggc att cc
Myostatin ttg aga ccc gtc gag act cct a ttc aga gat cgg att cca gta tac c
Myogenin gct gta tga gac atc ccc cta ctt cgt agc ctg gtg gtt cga a
Beclin-1 agg aac tca cag ctc cat tac aat ggc tcc tct cct gag tt
LC3 atg tca aca tga gcg agt tgg t ctg gtt cac cag cag gaa gaa

Definition of abbreviations: HMBS, hydroxymethylbilane synthetase; LC3, microtubule-associated protein 1A/1B-light chain 3;
MuRF, muscle-specific RING finger protein; Myf, myogenic factor; YWHAZ, tyrosine 3 monooxygenase/tryptophan 5 monoxygenase.

10 minutes at 48C. The supernatant (soluble fraction) was centrifuged RESULTS

further for 10 minutes at 48C and 10,000 3 g. The pellet (insoluble
fraction) was washed with PBS, resuspended, dissolved by sonication in Characterization of the COPD Patient and
200 ml buffer containing protease/phosphatase inhibitors (as described Control Populations
previously here), and further centrifuged (10,000 3 g, 48C) for
10 minutes, and the supernatant designated as the insoluble fraction. A total of 94 individuals with severe COPD were assessed for
Protein lysate (7.5 mg) was separated by SDS-PAGE, immunoblotted, study inclusion. Of these, 37 were not eligible based on
and bands detected using chemiluminescence generated by Super- exclusion criteria. Of the remaining 57 eligible patients, 9
Signal West Femto Chemiluminescent Substrate (Thermo Scientific, provided informed consent and took part in the study. Nine
Waltham, MA) acquired with a charge-coupled device camera (Bio- age-matched healthy control individuals provided informed
Rad VersaDoc, Hercules, CA), and quantified using Quantity One consent and took part in the study.
software (Bio-Rad). Primary antibodies used included: actin (1:200 To characterize our study populations, body composition,
dilution; Sigma-Aldrich, St. Louis MO); AKT; phospho-AKT (Ser pulmonary function, and functional capacity were assessed and
473); phospho-GSK3a/b (Ser 21/9); inhibitory kB (IkB)-a; phospho
data are provided in Table 2. As expected, individuals with
IkB-a (Ser 32/36); NF-kB p65; phospho-NF-kB p65 (Ser 536); p70 S6
kinase (1:1,000 dilution; all from Cell Signaling, Boston, MA); lamin
severe COPD had significantly lower 6-minute walk distances
A 1 C and MuRF1 (1:1,000 dilution; both from Abcam, Cambridge, and significantly higher BODE scores compared with control
MA); phosphop70S6 kinase (Thr 389, 1:1,000 dilution; Cell Signaling); individuals. There were no differences between the COPD and
MyoD (1:500 dilution; Santa Cruz, Santa Cruz, CA); GAPDH control populations with respect to BMI, percent body fat, or
(1:100,000 dilution; Abcam); GSK3a/b (1:10,000 dilution; Invitrogen, sum of five skin folds. The distribution of males and females was
Carlsbad, CA); and a-tubulin (1:50,000 dilution; Sigma). Secondary not equal between groups, with more females in the control
antibodies consisted of horseradish peroxidase (HRP)conjugated anti- group. Two male patients with COPD were involved in a pre
mouse, anti-rabbit, and anti-goat at 1:20,000 dilution. Phosphorylated lung transplant rehabilitation program, and one female patient
proteins were normalized in expression to their total unphosphorylated undertook regular aerobic and resistance training at least three
forms. MyoD, Nedd4, and MuRF1 were normalized to two house-
times per week. The remaining patients with COPD were all
keeping proteins, a-tubulin and GAPDH. Given the limited sample
available from the muscle biopsies, the levels of some proteins of fully independent and intermittently exercised. Four control
interest were not determined in all study participants. Total numbers participants (two male and two female) undertook regular
assessed are detailed in the results section. aerobic training greater than three times per week. The re-
mainder intermittently exercised.
Activated NF-kB(p65) and NF-kB(p50) Assays To confirm the presence of muscle atrophy in the patients
Quantitation of the expression of active NF-kB(p65) and active NF- with COPD, we measured CSA of the quadriceps femoris
kB(p50) was completed using the Thermo Scientific Transcription muscle. Patients with COPD demonstrated smaller quadriceps
Factor Kits for NF-kB(p65) and NF-kB(p50) (nos. 89858 and 89859; CSA (61.26 6 5.36 cm2; n 5 9) compared with control in-
ThermoFisher Scientific, Waltham, MA), as directed by the manufac- dividuals (89.88 6 4.59 cm2; n 5 9) as determined by computed
turer. Briefly, 7 mg of protein lysates were incubated with the NF-kB tomography imaging (P , 0.05; Figure 1A). Similarly, the
binding biotinylated consensus sequence DNA (p65 or p50) adherent strength of the quadriceps was lower in the patients with
to the streptavidin-coated 96-well plates provided. Only the active NF- COPD than the control subjects (11.95 6 1.06 kg; n 5 8 versus
kB (p65 or p50) binds to the consensus sequence, and is subsequently
detected with antiNF-kB(p65) or NF-kB(p50) primary antibody, an
HRP-conjugated secondary antibody and a chemiluminescent sub- TABLE 2. SUBJECT CHARACTERISTICS
strate (all provided in the kits). Chemiluminescence was detected in
Patient Subjects Control Subjects
an EnVision Multilabel Plate Reader Model 2102 (Perkin Elmer, Characteristics (n 5 9) (n 5 9) Significance
Waltham, MA) and reported as relative chemiluminescence units.
Specificity of the binding of the activated transcription factors to the Age, yr 64.0 6 2.1 59.6 6 1.3 N.S.
consensus sequence DNA was ensured by using control wild-type and Sex, male/female 5/4 3/6
mutant NF-kB (p65 or p50) competitor duplexes supplied with the kit. Body mass index 24.4 6 1.1 25.9 6 1.8 N.S.
Body fat, % 26.8 6 2.0 26.8 6 2.0 N.S.
Statistical Analysis Sum of five skin folds, cm 58.7 6 7.5 65.3 6 5.0 N.S.
Waist circumference, cm 92.7 6 3.0 99.8 6 11.9 N.S.
Continuous data are reported as mean and SE, and were compared using FEV1, L/s 0.88 6 0.09 2.87 6 0.22 P , 0.05
unpaired Students t test or Wilcoxons rank-sum test after testing for FEV1% predicated 35.1 6 2.5 115.1 6 7.9 P , 0.05
normal distribution (Shapiro-Wilk). When comparing multiple indepen- BODE 4.78 6 0.45 0.11 6 0.11 P , 0.05
dent means, a one-way ANOVA was first performed to confirm a 6-min walk, m 393 6 32 617 6 22 P , 0.05
difference across all groups prior to comparison of individuals means.
Results were considered significant if P values were less than 0.05. Definition of abbreviations: N.S., not significant.
464 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
23.46 6 3.02 kg, n 5 9; P , 0.05; Figure 1B). One male patient
declined strength testing.
Due to the uneven distribution of males and females in
the patient and control populations, we assessed differences
on a sex-specific basis. Female patients muscle area (46.37 6
5.95 cm2; n 5 4) was smaller than female control individuals
(81.59 6 2.92 cm2; n 5 6; P , 0.05; Figure 1C). Male patients
muscle area (73.18 6 1.55 cm2; n 5 5) was significantly smaller
than both male control subjects (106.50 6 1.94 cm2; n 5 3; P ,
0.05) and female control subjects (P , 0.05; Figure 1C). Sex-
specific comparisons were also performed for strength (Figure
1D). Female patients quadriceps strength was significantly
weaker than female control subjects (11.4 6 1.9 kg [n 5 4]
versus 20.1 6 2.6 kg [n 5 6]; P , 0.05). Male patients were
weaker than male control subjects (12.5 6 1.1 kg [n 5 4] versus
30.1 6 6.5 kg [n 5 3]; P , 0.05). Male patients, however, were
not significantly weaker than female control subjects (P 5 0.062).
Ubiquitin LigasesMediators of Protein Degradation by
the Proteasome
Good-quality RNA was extracted for all study participants.
Patients with COPD demonstrated a significant increase in the
level of atrogin-1 transcript expression (2.8 6 0.5fold change;
n 5 9) compared with the control individuals (1.4 6 0.2fold
change; n 5 9; P , 0.05) (Figure 2A). There was no difference
in the expression level of MuRF1 transcripts between patients
with COPD (10.7 6 2.1fold change; n 5 9) and control
individuals (8.8 6 2.2fold change; n 5 9; P . 0.05) (Figure
2B), nor was there any difference in MuRF1 proteins levels in
the soluble fraction of muscle protein lysates between patients
with COPD (0.22 6 0.10; n 5 9) and control individuals (0.11 6
0.03; n 5 9; P . 0.05) (Figures 2D and 2F).
In contrast, Nedd4 was increased in expression in the
insoluble fraction of muscle protein lysates (insoluble myofi-
brils) in the patients with COPD (0.37 6 0.08; n 5 9) versus
control subjects (0.09 6 0.03; n 5 9; P , 0.05) (Figures 2C and
2E). Nedd4 was also detectable in the soluble fraction of protein
lysates, but no differences in expression were seen in this
Figure 1. Quadriceps cross-sectional area (CSA)
and power. Patients with chronic obstructive
pulmonary disease (COPD) demonstrate signif-
icantly decreased CSA of the quadriceps femoris
muscle (A) and are weaker (B) than the age-
matched control subjects. Significant differ-
ences in muscle area between patients and
control subjects are maintained even when data
are assessed in sex-specific groups (C). Quadri-
ceps of female patients are significantly weaker
than in female control subjects (D), and male
patients are significantly weaker than male
control subjects, but male patients are not
significantly weaker than female control sub-
jects. F, female; M, male.
fraction between patients with COPD and control individuals
(data not shown).
Mediators of Muscle Regenerative CapacityMuscle
Regulatory Transcription Factors and Myostatin
Real-time RT-PCR was performed for myostatin, a negative
regulator of muscle growth and the muscle regulatory transcrip-
tion factors, myogenin and Myf5, which induce myoblast
differentiation and muscle regeneration. Myostatin transcript
levels (Figure 3A) were significantly higher in the patients with
COPD (3.1 6 0.8fold change; n 5 9) compared with the
control group (0.9 6 0.1fold change; n 5 9; P , 0.05). Neither
Myf5 (Figure 3B) nor myogenin (Figure 3C) demonstrated
significant differences in expression between patients with
COPD (n 5 9) and the control population (n 5 9) (Myf5,
0.96 6 0.18 versus 0.88 6 0.15fold change [P . 0.05], and
myogenin, 1.32 6 0.27 versus 0.96 6 0.16fold change [P .
0.05]). Similarly, there was no difference in the level of
expression of the proregenerative MyoD in the soluble fraction
of muscle protein lysates in patients with COPD (0.14 6 0.05;
n 5 8) versus control individuals (0.06 6 0.03; n 5 8; P . 0.05;
Figures 3D and 3E).
NF-kB Signaling NetworkInflammatory mediators
Phosphorylation of IkBa leads to its proteolytic degradation,
and subsequent release and activation of NF-kB transcription
factors. Total IkBa and phosphorylated IkBa (pIkBa) protein
levels (Figure 4) were determined in the soluble fraction of
muscle protein lysates by Western blotting. There was no
significant difference in the level of phosphorylation of IkBa
in the patients with COPD compared with the control popula-
tion (Figure 4), as determined by measuring the ratio of
phosphorylated IkBa/total IkBa (patients with COPD, 0.34 6
0.11 [n 5 9], versus control subjects, 0.22 6 0.09 [n 5 8]; P .
0.05) or when normalizing phosphorylated IkBa levels to
GAPDH (patients with COPD, 0.18 6 0.05 [n 5 9] versus
control subjects, 0.20 6 0.04 [n 5 8]; P . 0.05).
Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD 465

Figure 2. Ubiquitin ligases. Atrogin-1 mRNA levels (A) were increased in patients with chronic obstructive pulmonary disease (COPD) versus control
individuals, as determined by relative quantification with real-time RT-PCR. There was no difference in muscle-specific RING finger protein (MuRF) 1
transcript levels (B) between patients and control subjects. mRNA levels in patients and control subjects were compared with a common reference
standard. Tyrosine 3 monooxygenase/tryptophan 5 monoxygenase (YWHAZ) served as the housekeeping gene (HKG), and similar data were
obtained using hydroxymethylbilane synthetase (HMBS) as the HKG (data not shown). Neural precursor cellexpressed developmentally down-
regulated (Nedd) 4 protein levels (C ) were increased in the insoluble fraction of patient muscle protein lysates compared with control subjects, as
determined by SDS PAGE, Western blotting, and charge-coupled device camera image acquisition, and quantification of the chemiluminescent
signal. Nedd4 expression was normalized to a-tubulin, and a representative Western blot is shown (E ). Similar results were obtained normalizing
Nedd4 to GAPDH (data not shown). There was no difference in MuRF1 protein (D) expression in the soluble fraction of muscle protein lysates
between patients and control subjects. MuRF1 protein levels were normalized to a-tubulin, and a representative Western blot is shown (F ). Similar
results were obtained normalizing to GAPDH (data not shown). Co, control individual; N.S., not significant; Pt, patient; P . 0.05.

Quantitation of activation of NF-kB(p65) and NF-kB(p50)
was determined by measuring the chemiluminescence signal
generated by binding of the activated transcription factors to
their respective DNA consensus sequences fixed in a 96-well
plate and detected with antiNF-kB(p65) and antiNF-kB(p50)
primary antibodies and HRP-linked secondary antibodies. There
was no significant difference in the level of expression of active
NF-kB(p65) in patients with COPD (7.23 6 1.6 3 105 chem-
iluminescence units; n 5 6) versus control subjects (6.4 6 1.7 3
105 chemiluminescence units; n 5 6) (P . 0.05; Figure 5A).
Similarly, there was no difference in the level of expression of
active NF-kB(p50) between patients with COPD (1.9 6 0.8 3
106 chemiluminescence units; n 5 5) and control subjects (1.2 6
0.5 3 106 chemiluminescence units; n 5 6) (P . 0.05; Figure 5B).
AKT Signaling NetworkMediators of Muscle Hypertrophy
Total AKT and phosphorylated AKT, total GSK3b and phos-
phorylated GSK3b, and total p70S6 kinase and phosphorylated
p70S6 kinase protein levels were determined in the soluble
fraction of the muscle protein lysates by Western blot analysis.
There were no differences in the level of phosphorylation of
AKT (Figure 6) in the patients with COPD (0.44 6 0.13; n 5 8)
versus the control population (0.30 6 0.10; n 5 9) (P . 0.05), the
level of phosphorylation of GSK3b (Figure 7) in the patients with
COPD (0.58 6 0.16, n 5 8) versus the control population (0.74 6
0.17; n 5 9) (P . 0.05) or level of phosphorylation of p70S6
kinase (Figure 8) in the patients with COPD (0.34 6 0.10; n 5 8)
versus the control population (0.29 6 0.08; n 5 9) (P . 0.05).
Mediators of AutophagyBeclin-1 and LC3
There was no difference in the level of expression of beclin-1
transcripts in patients with COPD (6.7 6 1.2 fold change; n 5 9)
compared with control individuals (7.4 6 1.1 fold change; n 5 9)
(P . 0.05; Figure 9A). Similarly, there was no difference in the
level of expression of LC3 transcripts between patients with
COPD (52.4 6 16.5 fold change; n 5 9) and control individuals
(54.3 6 8.5 fold change; n 5 9) (P . 0.05; Figure 9B).
DISCUSSION
Skeletal muscle atrophy and weakness occur in individuals with
advanced COPD, and this reduction in muscle mass is associ-
466 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010

Figure 3. Muscle-regulatory transcription

factors and myostatin. Myostatin mRNA
levels (A) were significantly increased in
patients with chronic obstructive pulmonary
disease (COPD) compared with control sub-
jects, as determined by relative quantifica-
tion with real-time RT-PCR. There were no
differences in the level of expression of (B)
myogenic factor (Myf)5 transcripts or (C)
myogenin transcripts between patients with
COPD and control subjects. Transcript levels
in patients and control subjects were com-
pared with a common reference standard.
YWHAZ served as the HKG. Results were
similar using HMBS as the HKG (data not
shown). MyoD (D) protein levels in the
soluble fraction of muscle protein lysates
were not significantly different between
patients with COPD and control subjects,
as determined by SDS PAGE, Western blot-
ting, and charge-coupled device camera
image acquisition, and quantification of
the chemiluminescent signal. MyoD expres-
sion was normalized to a-tubulin. (E) A
representative Western blot is shown. Simi-
lar results were obtained normalizing MyoD
to GAPDH (data not shown). Co, control;
N.S., not significant; Pt, patient; P . 0.05.

ated with increased morbidity, mortality, health resource use, atrophy (reviewed in References 68) have been derived from
and cost (1, 35). The significant advances made in the past experimentation conducted largely in rodent models of disease,
decade delineating the cellular signaling pathways and key and the clinical relevance of these findings is not known. In
mediators responsible for the development of skeletal muscle the present study, we sought to identify the cellular signaling

Figure 4. Inhibitory kB (IkB)-a.

(A) There was no significant
difference in the level of IkBa
phosphorylation in the pa-
tients with chronic obstructive
pulmonary disease (COPD)
versus control individuals. To-
tal and phosphorylated IkBa
(IkBa and pIkBa) protein
levels were determined in the
soluble cytoplasmic fraction of
muscle protein lysates by SDS-
PAGE, Western blotting, and
charge-coupled device camera
image acquisition, and quan-
tification of the chemilumines-
cent signal. pIkBa expression
was normalized to total IkBa
and to GAPDH. (B) A repre-
sentative Western blot is
shown. Positive (Pos) and neg-
ative (Neg) controls for IkB
phosphorylation consisted of
protein lysates from HeLa cell
cultures treated with TNF-a
(20 ng/ml 3 5 min) and un-
treated, respectively. Co, con-
trol; N.S., not significant; Pt,
patient; P . 0.05.
Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD
Figure 5. NF-kB(p65) and NF-kB(p50) activation. Expression levels of
the active forms of NF-kB(p65) and NF-kB(p50) proteins were de-
termined in muscle protein lysates using the ThermoScientific Transcrip-
tion Factor Kits for NF-kB(p65) and NF-kB(p50). Activated p65 and p50
bound to the NF-kBbinding consensus sequence DNA immobilized in
96-well plates was detected using antiNF-kB(p65) and NF-kB(p50)
antibodies, horseradish peroxidaselinked secondary antibodies and
a chemiluminescent substrate. The chemiluminescent signal was quan-
tified in an EnVision multilabel plate reader. There was no significant
difference in the level of expression of active NF-kB(p65) (A) or
NF-kB(p50) (B) in the patients with chronic obstructive pulmonary
disease (COPD) versus control individuals. N.S., not significant; P .
0.05 (59, 60).
networks responsible for the development of muscle atrophy in
individuals with COPD. Our patients with COPD were care-
fully selected to exclude comorbid illness and medical therapies
that could affect muscle mass. Therefore, the significant atrophy
and weakness of the quadriceps femoris muscle demonstrated
most likely results from the advanced but stable COPD in our
patient population. We report the novel observations of up-
regulation of myostatin and the ubiquitin ligase, Nedd4, and
confirm up-regulation of a second ubiquitin ligase, atrogin-1
(which has been previously reported [37]), in the atrophied
vastus lateralis muscle of individuals with severe COPD.
Skeletal muscle protein loss is mediated by a series of
intracellular signaling networks, but ubiquitin-dependent pro-
teolytic mechanisms are the predominant mechanisms respon-
sible for the development of muscle atrophy (reviewed in
References 6, 812). Protein ubiquitination is a highly ordered
process whereby proteins to be degraded are tagged with
multiple ubiquitin moieties, which serve as recognition markers,
targeting proteins for subsequent proteolytic cleavage by the
26S proteasome or lysosome. Ubiquitin ligases are the critical
enzymes that both link ubiquitin moieties to the protein
targeted for degradation and confer specificity to the system
by interacting directly with the target protein through well-
defined proteinprotein interaction domains (38). The ubiquitin
ligases, atrogin-1 and MuRF1, are key regulators of muscle
atrophy in multiple rodent models (14, 15, 17), and, more
467
Figure 6. AKT. (A) There was no difference in the level of phosphory-
lation of AKT in the patients with chronic obstructive pulmonary
disease (COPD) versus the control population. Total AKT and phos-
phorylated AKT (pAKT) protein levels were determined by SDS-PAGE,
Western blotting, charge-coupled device camera image acquisition,
and quantitation of the chemiluminescent signal in the soluble fraction
of muscle protein lysates. pAKT protein levels were normalized to total
AKT protein levels. (B) A representative Western blot is shown. Protein
lysates of 293T cell cultures treated with insulin, and untreated, served
as positive (Pos) and negative (Neg) controls, respectively, for AKT
phosphorylation. Western blotting for GAPDH served as a loading
control. Co, control; N.S., not significant; Pt, patient; P . 0.05.
recently, a third ligase, Nedd4, has also been recognized (13,
16, 39) to contribute to this process in rodent models.
The up-regulation of atrogin-1 and Nedd4 noted in our
patients with COPD infers an increase in ubiquitinproteasome
mediated proteolysis in the COPD muscle. Interestingly we did
not find MuRF1 to be increased. Discordance in the up-
regulation of these three ubiquitin ligases has been previously
reported, with the pattern and temporality of ligase recruitment
dependent upon the etiology of the muscle atrophy. Ottenheijm
and colleagues (40) reported enhanced proteasomal degrada-
tion in the diaphragms of patients with COPD, and found
increased transcript levels of atrogin-1, but not MuRF1, in the
diaphragmatic muscle. Nedd4 expression was not investigated
in this population. Nedd4 is increased in atrophy associated with
denervation or unloading (8, 13, 41), but is not increased in
muscle atrophy resulting from diabetes, renal failure, or star-
vation in mice or rats (16, 17, 39). Atrophy resulting from
denervation in rodents involves the up-regulation of atrogin-1,
MuRF1, and Nedd4, but atrogin-1 and MuRF1 increases are
short lived, with persistent increases seen only in the level of
Nedd4 (13). Thus, there is specificity of these ubiquitin ligases
to the systemic process responsible for the development of
atrophy and in the temporality of its progression. This is not
surprising, as the target substrates identified to date for each
enzyme differ (13, 16, 4244). Clearly, in the human population,
further study is necessary to fully understand the implications of
this specificity in muscle atrophy arising from different disease
processes. Our novel finding that Nedd4 is up-regulated, and
confirmation of atrogin-1 up-regulation in the COPD patient
population, is significant, as it provides possible targets for
future therapeutic intervention in the treatment of COPD.
It is interesting that we note an increase in Nedd4 in the
muscle of patients with COPD, whereas up-regulation of Nedd4
in rodent models of atrophy has been limited to unloaded or
468 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010

Figure 7. Glycogen synthase kinase (GSK)3b. (A) There was

no significant difference in the level of phosphorylation of
GSK3b in the patients with chronic obstructive pulmonary
disease (COPD) versus the control population. Total GSK3b
and phosphorylated GSK3b (pGSK3b) protein levels in the
soluble fractions of muscle protein lysates were determined
by SDS-PAGE, Western blotting, charge-coupled device cam-
era image acquisition, and quantification of the chemilumi-
nescent signal. pGSK3b protein levels were normalized to
total GSK3b levels. (B) A representative Western blot is
shown. Protein lysates of 293T cell cultures treated with
insulin, and untreated, served as positive (Pos) and negative
(Neg) controls, respectively, for GSK3b phosphorylation.
Western blotting for GAPDH served as a loading control.
Co, control; N.S., not significant; Pt, patient; P . 0.05.

denervated muscle. The unloading model in rats (hindlimb deletion of the myostatin gene demonstrate a hypermuscular
suspension) is an extreme muscle disuse model. It may be that phenotype (27). Increased myostatin expression in both serum
there is a graded effect of muscle loading on Nedd4 expression, and muscle was associated with decreased muscle mass in HIV-
with relative muscle disuse resulting in only slightly increased positive patients with muscle wasting (49).
Nedd4 expression. This would not have been noted in the rat Our finding of increased levels of myostatin transcripts in the
models of diabetes, renal failure, or starvation, because these patients with COPD implies that the loss of muscle mass in our
animals retain full activity. Our patients with COPD were COPD population is attributed to impaired muscle growth, in
ambulatory; their muscles are loaded and innervated, but they addition to active muscle degradation, reiterating the notion
may experience relative disuse compared with control individ- that atrophy is a multifactorial process rather than the result of
uals, resulting in increased Nedd4 expression. An alternative a single biochemical pathway/signal. We cannot comment on
explanation is that we noted increased Nedd4 expression only in the signaling pathways engaged downstream of myostatin, as we
the insoluble myofibrillar fraction of protein lysates, not in the did not interrogate the cell cycle regulators. However, as we did
soluble fraction. Koncarevic and colleagues (16) localized not demonstrate any changes in the levels of MyoD, myogenin,
Nedd4 to the sarcolemma in healthy rodent muscle, and
assessed levels only in cytoplasmic and crude membrane
fractions. We have noted increased expression of Nedd4 in
the insoluble myofibrillar fraction of protein lysates of rodent
muscle atrophying from denervation (39). In other organs and
cells, Nedd4 is expressed throughout the cytoplasm, with
membrane localization occurring only upon target substrate
binding (45, 46). Given the varying subcellular distribution, and
the fact that the downstream target substrates of Nedd4 in
muscle remain largely undefined, it is not possible to state in
which fraction increased Nedd4 expression is necessary for the
development of muscle atrophy.
In addition to enhanced protein breakdown, alterations in
the regeneration of muscle can influence muscle mass. Muscle
satellite cells are the population of precursor cells that re-
generate muscle and maintain muscle mass (47, 48). These cells
are normally quiescent, but they are activated to proliferate
after muscle injury, terminally differentiating to mature myo-
cytes, and fusing to form new myofibers or repair damaged
myofibers. The MRFs, MyoD, Myf5, and myogenin, are essen-
tial to the processes of muscle self-renewal, and specify satellite
Figure 8. p70S6 Kinase. (A) There was no significant difference in the
cell terminal differentiation into muscle cells. We found no
level of phosphorylation of p70S6 kinase in the soluble fraction of
differences in the level of expression of Myf5, myogenin, or
muscle protein lysates of patients with chronic obstructive pulmonary
MyoD between patients and control subjects, suggesting that
disease (COPD) versus the control population. Total p70S6 kinase and
myoblast differentiation in COPD muscles is unaffected. phosphorylated p70S6 kinase (p-p70S6 kinase) protein levels were
Myostatin is a member of the transforming growth factor b determined by SDS-PAGE, Western blotting, charge-coupled device
family, and is a negative regulator of skeletal muscle develop- camera image acquisition, and quantification of the chemiluminescent
ment and growth. Myostatin inhibits proliferation of satellite signal. p-p70S6kinase expression was normalized to total p70S6 kinase
cells/myoblasts by up-regulating expression of p21 and decreas- expression. (B) A representative Western blot is shown. Protein lysates
ing Cdk2 and phosphorylated Rb, which results in cell cycle of 293T cell cultures treated with insulin, and untreated, served as
withdrawal (27). Myostatin also appears to inhibit satellite cell positive (Pos) and negative (Neg) controls, respectively, for p70S6
differentiation via the down-regulation of various MRFs, in- kinase phosphorylation. Western blotting for GAPDH served as a load-
cluding MyoD, Myf5, and myogenin. Mice with a targeted ing control. Co, control; N.S., not significant; Pt, patient; P . 0.05.
Plant, Brooks, Faughnan, et al.: Markers of Muscle Atrophy in COPD
Figure 9. Autophagy factors beclin-1 and microtubule-associated pro-
tein 1A/1B-light chain 3 (LC3). There was no difference in the level of
expression of beclin-1 or LC3 transcripts between patients with chronic
obstructive pulmonary disease (COPD) and control individuals, as
determined by relative quantification with real-time RT-PCR. mRNA
levels in patients and control subjects were compared with a common
reference standard. YWHAZ served as the HKG, and similar data were
obtained using HMBS as HKG (data not shown). N.S., not significant;
P . 0.05.
or Myf5, it is probable that myostatin is affecting proliferation
of the satellite cell population as opposed to differentiation.
This would impair the regenerative capacity of COPD muscle,
and potentially contribute to the loss of muscle mass.
The NF-kB signaling pathway is known to mediate muscle
atrophy in rodent models of muscle wasting associated with
cachexia, systemic inflammation, denervation, and unloading (7,
8, 20, 23, 50) through the combined effects of stimulated
proteolysis and impaired muscle growth. Of the five known
transcription factors (p65, Rel B, C-rel, p52, and p50), p65 is the
primary subunit known to regulate muscle biology. As with all
NF-kB transcription factors, p65 heterodimers are held in the
cytosol in an inactive complex with IkB (24, 51, 52). p65
activation occurs via phosphorylation of IkBa, release, and
translocation to the nucleus. Phosphorylation of NF-kB(p65) in
the nucleus enhances its transcriptional activity. It is there that
the p65/p50 heterodimers bind the MuRF1 promotor, inducing
MuRF1 transcription and proteasomal mediated muscle degra-
dation (23). p65 is the sole subunit currently shown to regulate
myogenesis (24, 53).
There are reports suggesting that up-regulation of NF-kB
signaling is involved in the development of muscle atrophy
associated with COPD. Agusti and colleagues (50) demon-
strated NF-kB(p65) activation in pooled muscle protein lysates
from patients with COPD with low BMI, compared with
patients with COPD with normal to high BMI. Inflammatory
cytokines, such as TNF-a, induce muscle atrophy by engaging
NF-kB(p65) signaling, which results in increased proteasomal-
mediated protein degradation and inhibition of muscle regen-
erative pathways via the induction of oxidative stress, and
decreased MyoD expression (20, 22). Low-grade systemic in-
flammation (elevated circulating plasma levels of TNF-a, and
IL-1 and -6) has been described in patients with COPD (54, 55).
Despite these reports of up-regulation of systemic inflammation
in patients with COPD at both a resting state and during and that by Doucet and colleagues may be attributable to
acute exacerbations, and in contrast to Agustis findings, we
were unable to demonstrate engagement of IkBa or NF-
kB(p65) in the vastus lateralis muscles of our patients with
COPD. The absence of engagement of NF-kB(p65) signaling in
our patients is supported by normal expression levels of MuRF1
and MyoD, both downstream of p65. In contrast to Agustis
patient cohort, our patient population had a normal BMI, which
may have contributed to the difference in findings between our
studies.
469
Other investigations have recently addressed the question
of the presence of a proinflammatory milieu in the skeletal
muscle of stable patients with COPD compared with healthy
control subjects. Neither Barreiro and colleagues (56) nor Crul
and colleagues (57) were able to demonstrate local inflamma-
tion in the vastus lateralis muscle of clinically and weight-
stable patients with COPD by assessing the level of expression
of various cytokines. Barreiro did, however, demonstrate
increased oxidative stress in the COPD muscle. Thus, studies
performed in humans to date, including our results here,
suggest that inflammatory mediators and signaling via NF-
kB(p65) are not active in the muscle of clinically stable
patients with COPD, and do not contribute to the develop-
ment of muscle atrophy in those with normal weight and BMI.
This does not preclude the possibility that, during acute illness,
this pathway may be recruited, but our study did not address
this issue.
Recently, NF-kB(p50) has been shown to mediate muscle
atrophy associated with unloading, independent of activation of
p65 (58). The downstream substrate targets of p50 that mediate
this effect are unknown, and, to date, no other upstream
stimulants that engage this atrophic pathway have been identi-
fied. We interrogated this pathway here, because muscle
deconditioning and relative disuse can occur in the COPD
population. However, it appears that, in the patient with stable
COPD, p50 engagement does not contribute to muscle atrophy.
Whether there is an incremental effect, with p50 recruitment
during acute exacerbations of COPD in a comparable patient
population, remains to be determined.
Phosphorylation and activation of AKT is well known to
induce skeletal muscle hypertrophy (6, 25). AKT activation
results in the recruitment and subsequent phosphorylation of
multiple direct and indirect downstream targets, including
mammalian target of rapamycin, p70S6 kinase, PHAS-1, and/
or GSK3b, all of which contribute to the development of muscle
hypertrophy, at least in part, via the induction of protein
synthesis. It has also been recently appreciated that activation
of AKT signaling suppresses the expression of atrogin-1 and
MuRF1 via phosphorylation of the Forkhead O transcription
factors, and thereby actively suppresses muscle proteolysis (25,
26). Conversely, down-regulation of AKT signaling occurs in
muscle atrophied by denervation, disuse, and glucocorticoid
administration, demonstrating reciprocal communication be-
tween pathways mediating muscle atrophy and hypertrophy
(8). The amount of material available to us from the muscle
biopsy limited the extent to which we were able to interrogate
the AKT signaling network, and we focused our assessment on
AKT, GSK3b, and p70S6 kinase. Surprisingly, despite the
atrophy seen in the COPD population, we were unable to
demonstrate down-regulation of AKT signaling in our cohort of
patients with COPD.
In contrast, Doucet and colleagues (37) demonstrated an
increase in protein levels of phosphorylated AKT, GSK3b, and
p70S6 kinase in patients with COPD, and hypothesized a com-
pensatory mechanism of muscle mass recovery accounting for
this activation (37). The discrepant findings between our study
differences employed in the normalization of data, yet the
critical observation is that neither study found AKT signaling
to be down-regulated in the patients with COPD. This was
unexpected and in contrast to data generated in rodent models
of muscle atrophy. This finding suggests the possibility that
a compensatory response is in place in the muscle of patients
with COPD that attempts to counteract the atrophic stimuli.
There is certainly precedent for this phenomenon. Attempts at
muscle recovery after muscle atrophy are described in other
470 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010
models (59), but this recovery response is temporally limited,
and can be exhausted.
Activation of autophagy has recently been shown to con-
tribute to the development of muscle atrophy. LC3 and beclin-1,
both known components of autophagy signaling, have been
shown to increase in rodent models of denervation and unloading-
induced muscle atrophy (18). In C2C12 myotubes in vitro,
autophagy has been demonstrated to play as important a role
as ubiquitinproteasomemediated muscle proteolysis (19). In
addition, AKT signaling has been shown to be reciprocally
linked to autophagy, as it has been to ubiquitin-mediated deg-
radation (60, 61). Decreased AKT signaling results in relative
dephosphorylation and nuclear translocation of Forkhead O
transcription factors, which, in turn, results in the increased
expression of multiple autophagy-related genes and the in-
duction of autophagy. Regardless of the mounting evidence
detailing the importance of autophagy in skeletal muscle
atrophy, we did not note an increase in beclin-1 or LC3
transcript levels in our COPD population.
Although our study carefully describes molecular changes
seen in muscle atrophy associated with stable COPD compared
with that in control subjects, there are some limitations inherent
in clinical molecular studies. First, we were unable to identically
match our control and COPD populations, with more females in
the control group. However, to date, we are unaware of data
that support pathways inducing muscle atrophy that are differ-
entially regulated in males and females. Unlike rodent studies,
limited clinical material requires a selective, rather than ex-
haustive, evaluation of molecular pathways, and the total
number of participants was small. There is a risk that the study
is underpowered as a result, and further work is required to
complement our study. Finally, a dearth of reliable antibodies
for specific targets of interest, combined with limited clinical
material, restricts some of the data to transcriptional mRNA
expression only, but we believe that this is reflective of total
cellular protein levels for the mediators that we assessed.
Thus, we have demonstrated, in a well-characterized cohort
of individuals with severe COPD and decreased skeletal muscle
mass, the up-regulation of mediators of protein degradation
(atrogin-1 and Nedd4) and of the myogenic inhibitory factor,
myostatin, in the vastus lateralis muscle. These findings imply
that the muscle atrophy associated with COPD results from
both an inhibition of the reparative capacity of muscle and
increased ubiquitin-mediated proteolysis, and that there is
specificity in the ubiquitin ligases recruited to the etiology of
muscle atrophy in humans. Whether the changes in the biology
of the muscle in the population of patients with COPD results
from the lung disease itself, or the combination of a chronic
disease accompanied by possible altered nutritional status and/
or diminished activity, is not clear. Regardless, characterizing
the biological networks activated in the atrophied muscle of
humans with end-stage COPD is necessary to allow the de-
velopment of therapeutic interventions to counteract muscle
atrophy and to improve patient function, quality of life, and,
potentially, survival.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank Forough Farrokhyar for her assistance with
the statistical analysis.
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